Enhanced production of D-psicose from D-fructose by a redox-driven multi-enzyme cascade system.

D-Psicose, a new-generation sugar substitute, has been enzymatically synthesized through D-fructose isomerization. However, isomerization often causes low yields due to unfavorable thermodynamic equilibria, which limited its further industrial application. In this study, we present a redox-driven multi-enzyme cascade, two-step biotransformation system to produce D-psicose from D-fructose. Compared to D-fructose isomerization, this method has a maximized theoretical conversion rate of 100%. D-Psicose-3-epimerase from Clostridiales (CBDPE), ribitol 2-dehydrogenase from Providencia alcalifaciens (PRDH), and formate dehydrogenase from Starkeya (SFDH) were co-expressed in Escherichia coli in the first step to produce D-allitol from D-fructose. Afterward, NADH oxidase from Streptococcus pyogenes (SPNOX), and ribitol 2-dehydrogenase from Rubrivivax sp. (RSRDH) were co-expressed in E. coli to oxidize D-allitol into D-psicose in the second step. The two-step biotransformation system was optimized to maximize the D-fructose-to-D-psicose conversion rate (up to 90%), corresponding to a concentration of 450 mM. This study suggests that this redox-driven multi-enzyme cascade strategy through a sugar-to-alcohol-to-sugar pathway has the advantage of great application for enhanced production of D-psicose and other rare sugars.